Figure 7. Actin associates with PPP1R15B to alter the level of eIF2α phosphorylation.
(A) Immunoblot for GFP and actin of HEK293T cell lysates expressing either GFP or GFP-PPP1R15B. Upper two panels indicate proteins immunoprecipitated by anti-GFP beads. Lower panel shows 2% of input lysate. (B) Immunoblot for P-eIF2α, total eIF2α, and PPP1R15A of lysates from WT or Ppp1r15btm1Dron/tm1Dron MEFs treated for 1 hr with thapsigargin 400 nM, jasplakinolide 1 µM or both. (C) Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α and actin. Ppp1r15atm1Dron/tm1Dron MEFs were treated with jasplakinolide 1 µM for the indicated times. Lysates were subjected to sedimentation assay and immunoblot for G-actin in the supernatant (G) or F-actin in the pellet (F). (D) Immunoblot for phosphorylated eIF2α (P-eIF2α), total eIF2α, and actin. Ppp1r15atm1Dron/tm1Dron MEFs were treated with the indicated concentrations of jasplakinolide for 1 hr. Lysates were analysed as in ‘C’. Accompanying graphs show mean ± SEM of n = 3 independent repeats.